The rapidly activating delayed rectifier K- current (IKr), and the human ether-a-go-related gene (HERG) thought to encode it, play a key role in cardiac repolarization, and mutations in HERG cause congenital long QT syndrome (LQT-2). HERG, IKr and LQT-2 will be studied using molecular and electrophysiological techniques in transfected HEK293 cells and native rabbit myocytes. Specific aim 1 is to study cellular mechanisms of ion channel processing of HERG wild type and LQT-2 mutant channels. We have previously identified normal steps in the processing mechanism for HERG protein as well as abnormal processing for some LQT-2 mutants. We will extend these observations by testing three hypotheses: a) the failure of some LQT-2 mutants to function normally involves defects in multiple processing steps, b) co-expression of the minK or minK-related subunits modifies HERG wild type and LQT-2 protein trafficking, c) co-expression of wild type HERG protein with minK LQT mutations (LQT-5) alters the expression of HERG current. We will identify the steps where this occurs. Specific aim 2 is to study cell processes that modify HERG protein production and degradation. We will test the hypothesis that LQT-2 mutant proteins are degraded rapidly, compared with wild type HERG protein, which is of particular importance for newly described LQT-2 mutant channels that form functional channels. Such an increased turnover, if demonstrated, would be a novel mechanism for the expression of the LQT-2 phenotype. We will also test the hypothesis that N- linked glycosylation is a determinant of the stability of expressed HERG protein. We will test the effects of temperature as a determinant of the success and efficiency of trafficking of LQT-2 mutant channels to the surface membrane, and we will identify additional cell processes that might modify HERG protein trafficking. Specific aim 3 is to study the mechanisms of co- assembly of wild type HERG with LQT-2 mutant channel subunits. We will test the hypothesis that protein trafficking abnormalities alter expression of the dominant negative effect. We will test what role, if any, minK, minK-related, and minK mutant (LQT-5) channels have in modifying this. This research will increase our knowledge of molecular mechanisms of ion channel processing and function of HERG and more generally will have implications for all ion channels. More specifically it will have particular relevance to mechanisms of the human disease LQT-2. Elucidating mechanisms in these areas in important in developing new strategies for understanding normal and abnormal arrhythmogenesis and for new strategies for anti-arrhythmic therapies.